Effects of high glucose and K+ depolarization on submembrane granule turnover in primary beta-cells as visualized by Insulin-EGFP and C-peptide-emGFP

Background and aims:

The conventional view that insulin granules sequentially approach the plasma membrane, then bind and finally fuse when [Ca²⁺]_i increases has been challenged by the recognition that granules continually arrive at and depart from the submembrane space. Here it was tested whether the depolarization by high K⁺ elicits the same functional consequence as high glucose. To minimize the influence of labeling, two different cargo-directed labels were used.

Methods:

All parameters were measured in perifused cultured mouse beta-cells. [Ca²⁺]_i was measured by the Fura technique, the mobility of the granules (labeled by adenoviral transduction with Insulin-EGFP or C-peptide-emGFP) was imaged by TIRF microscopy. The temperature was 32 °C throughout.

Results:

40 mM KCl produced a fast and strong increase in [Ca²⁺]_i with an initial overshoot. 30 mM glucose led to a lower but more sustained elevation. Cell culture duration, but not the viral transduction as such, diminished the [Ca²⁺]_i response, with KCl being the more robust stimulus. KCl increased the total number of granules identified per imaging sequence, the number granules arriving at the submembrane space and the number of short term-resident granules. Glucose, when applied first, decreased these values, but had no clear-cut effect when it followed KCl. In this situation the results obtained with insulin-EGFP differed from those obtained with c-peptide-emGFP.

Conclusion:

While the behavior of granules labeled by insulin-EGFP and those labeled by c-peptide-emGFP was not exactly identical, the overall mean values suggest that K⁺ depolarization accelerates granule turnover whereas glucose diminishes it.